This work is aimed at exploring regulatory mechanisms involved in normal differentiation and malignant transformation, using the B16 melanoma model system involving melanotic and amelanotic malignant clones (and amelanotic nonmalignant clones derived from the melanotic clone after growth and maintenance in the presence of 1 microgram of 5-bromodeoxyuridine [BrdUrd] per ml). The BrdUrd clone C3471 can immunize syngeneic mice against the parental tumor. We are studying the leukocyte subpopulations elicited by immunization with BrdUrd clone C3471. Macrophages, T cells and possibly natural killer cells cooperate in rejection of the tumorigenic parental melanoma clone (B559). The role of interferon and other lymphokines and the part played by the endogenous virus induced by the presence of BrdUrd in the medium in which C3471 cells are grown are being determined. In assessing the role of the virus in the tumor rejection process, we found that injection of C3471 virus-infected feral mouse embryo cells (SC1) will protect mice against the malignant melanoma clone B559, whereas uninfected SC1 cells will not. Since preliminary studies indicated that cells infected with or spontaneously producing other B-tropic retroviruses were not immunogenic, we are planning to determine whether these viruses are detectably different at the level of genomic structure, as studied with partially purified or cloned intermediates. We have cloned several Thy-1.2-positive T-lymphocyte lines from the peritoneal exudate and spleens of C3471-immune mice. One lymphocyte clone bears the Lyt-2 antigen, is not cytotoxic, but in a Winn assay facilitates growth of coinjected melanoma cells. We are doing additional tests to determine whether this is a suppressor T-lymphocyte clone. In order to facilitate the study of the genetic and molecular basis of differentiation, we have been using calcium phosphate coprecipitation to cotransform cellular DNAs for specific differentiated function, and plasmid pSV2gpt, in order to make possible selection of markers such as pigment or hormone production in recipient melanoma clones. Some of these clones have proved to be more competent as recipients than most other murine cell lines. These approaches should lead to further understanding of regulatory mechanisms in malignancy, immunogenicity, melanogenesis and other differentiated functions.